According to most recent World Health Organization (WHO) estimates, around 170–200 million individuals have chronic HCV infection worldwide. The HCV prevalence shows significant geographic variations and demographic variations within a geographic area. In Europe, and particularly in the Mediterranean countries, the prevalence of HCV infection increases in parallel with age, while in the United States it is most common in persons 30–49 years of age, see W. R. Kim, The burden of hepatitis C in the United States, Hepatology 36 (2002) S30–S34. The incidence of new infections with HCV is declining in the developed countries while the number of new cases is still increasing in the underdeveloped countries, largely due to the use of contaminated blood for transfusion. The major risk factor for HCV transmission has changed over time from blood transfusion related cases to injecting drug use in the Western world. The relative importance of other risk factors has not changed much over time. These include unsafe sex with multiple partners, occupational and perinatal exposures, nosocomial and iatrogenic infections, unsafe tattooing, piercing and acupuncture, see M. J. Alter, Prevention of spread of hepatitis C, Hepatology 36 (2002) S93–S98.
Hepatitis C is caused by a small RNA virus belonging to the flaviviridae family and has been recently classified as the only member of the genes hepacivirus, see B. Robertson, G. Myers, C. Howard, T. Brettin, J. Bukh, B. Gaschen, et al., Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization, Arch Virol 143 (1998) 2493–2503. The HCV genome is a 9.6 Kb single-stranded RNA which encodes a single polypeptide of about 3000 amino acids, see M. Major, S. M. Feinstone, The molecular biology of hepatitis C, Hepatology 25 (1997) 1527–1538. This HCV polypeptide is cut post-translationally to generate several structural and non-structural proteins including two envelope glycoproteins (E1 and E2), the nucleopeptide protein (core-C) and several non-structural (from NS2 to NS5) proteins. Some of the viral proteins have been shown to involve in the pathogenesis of the liver disease and also in the development of resistance to interferon therapy. The HCV core proteins, either in its full-length or truncated forms, have been shown to provoke apoptosis of infected cells, see A. Ruggieri, T. Harada, Y. Matsuura, T. Miyamura, Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein, Virology 229 (1997) 68–76, and thus might directly involved in the pathogenesis of liver disease, of cell proliferation and liver cancer development. The core proteins and NS5A have also been reported to interfere with cellular metabolism of lipids and with a direct effect on the development of steatosis, see G. Perlemuter, A. Sabile, P. Letteron, G. Vona, A. Topilco, Y. Chretien, et al., Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis, FASEB J 16 (2002) 185–194, which is a characteristic feature of hepatitis C, see L. Rubbia-Brandt, R. Quadri, K. Abid, E. Giostra, P. J. Male, G. Mentha, et al., Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3, J Hepatol 33 (2000) 106–115. In addition, NS5A may contain an interferon sensitivity determining region (ISDR) capable of regulating the cellular response to interferon, see N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, et al., Mutations in the non-structural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection, N Engl J. Med. 334 (1996) 77–81. This protein region can bind and inhibit protein kinase R (PKR), whose activity is pivotal for the development of intracellular antiviral state in response to interferon, see S. L. Tan, M. G. Katze, How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A, Virology 284 (2001) 1–12.
There are six major different HCV genotypes and multiple subtypes. Genotypes 1a and 2b are most common in Europe and the United States, followed by genotypes 2 and 3. These four genotypes are also common in the rest of the world. On the other hand, the other genotypes are only common in particular geographic area, such as Egypt in the case of genotype 4, South Africa in the case of genotype 5, and Southeast Asia in the case of genotype 6. The viral genotype is important in terms of the treatment efficacy of antiviral therapy, see T. Poynard, P. Marcellin, S. S. Lee, et al., Randomized trial of interferon alpha 2b plus ribavrin for 48 weeks or for 24 weeks versus interferon alpha 2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 352 (1998) 1426–32, and J. G. McHutchison, S. C. Gordon, E. R. Schiff, et al., Interferon alpha-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J. Med. 339 (1998) 1485–92, with better responses associated with genotypes 2 and 3 than with genotype 1. Some of the HCV strains had been reported to have enhanced virulence, but the molecular determinants and mechanisms conferring this property remain elusive, see P. Farci, S. J. Munoz, A. Shimoda, et al., Experimental transmission of hepatitis C virus-associated fulminant hepatitis to a chimpazee. J Infect Dis 179 (1999) 1007–11. Furthermore, genetic variations within a region of NS5A have been deduced to associate with the treatment effectiveness of interferon therapy, as shown in isolates of Japanese subtype 1b, see N. Enomoto, I. Shakuma, Y. Asahina, et al., Mutation in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis virus 1b infection. N Engl J. Med. 334 (1996) 77–81. However, this result could not be reproduced in European and American isolates of HCV 1, see R. T. Chung, A. Monto, J. L. Dienstag, L. M. Kaplan, Mutations in the NS5A region do not predict interferon-responsiveness in American patients infected with genotype 1b hepatitis C virus. J Med Virol 58 (1999) 353–8, and S. Zeuzem, J. H. Lee, W. K. Roth, Mutations in the non-structural 5A gene of European hepatitis C virus isolates and response to interferon alpha. Hepatology 25 (1997) 740–4.
The natural history of hepatitis C is very heterogeneous, it can either progress towards cirrhosis and its complications, though over a quite long period of time, or remain as benign and one-progressive chronic infection in the majority of the HCV carriers. The severity, progression, and outcome of hepatitis are influenced by several cofactors, see A. Alberti, L. Chemello, L. Benvegnu, Natural history of hepatitis C, J Hepatol 31 (Suppl 1) (1999) 17–24. Retrospective studies conducted in patients with hepatitis C observed for 10–30 years after infection indicate that 17–55% (mean 42%) developed cirrhosis, 1–23% developed HCC and 4–15% died of liver related causes. These figures are quite reduced in most prospective studies where over a follow-up period of 8–16 years after exposure 7–16% of the patients developed cirrhosis (mean 11%), 0.7–1.3% developed HCC and 1.3–3.7% died of liver related causes, see L. B. Seeff, Natural history of chronic hepatitis C, Hepatology 36 (2002) S35–S46. In a series of retrospective-prospective studies lasting 9–45 years, it was found that 0.3–15% of the developed cirrhosis, 0–1.9% HCC, and 0–2.8% died of liver related diseases. These investigations also revealed that many host and environmental factors can influence the course and outcome of chronic hepatitis C and account for the great heterogeneity of this disease. These differences are very well described by the quite different outcomes and rates of progression to cirrhosis seen when distinct cohort of patients were followed-up for a similar period of time (20–25 years) after infection. In adult patients, mainly males, infected at the age of 45–65 years with a large inoculum through blood transfusion in the pre-serologic era, 15–27% developed cirrhosis, see R. L. Koretz, H. Abbey, E. Coleman, G. Gitnick, NANB post-transfusion hepatitis: looking back on the second decade, Ann Intern Med 119 (1993) 110–115, F. Tremolada, C. Cassin, A. Alberti, C. Drago, A. Tagger, M. L. Ribero, G. Realdi, Long-term follow-up of NANB (type C) post-transfusion hepatitis, J Hepatol 16 (1992) 273–281, and A. M. Di Bisceglie, Z. D. Goodman, K. G. Ishak, J. H. Hoofnagle, J. J. Melpolder, H. J. Alter, Long-term clinical and histopathological follow-up of chronic post-transfusion hepatitis, Hepatology 14 (1991) 969–974, compared to 4% with community-acquired hepatitis C, see A. J. Rodger, S. Roberts, A. Lanigan, S. Bowden, N. Crofts, Assessment of long-term outcomes of community-acquired hepatitis C infection in a cohort with sera stored from 1971–1975, Hepatology 32 (2000) 582–587, 1% of young drug-addicts, see D. L. Thomas, J. Astemborski, R. M. Rai, F. A. Anania, M. Schaeffer, N. Galai, et al., The natural history of hepatitis C virus infection: host, viral and environmental factors, J Am Med Assoc 284 (2000) 450–456, 0.4–2% of young women contaminated by anti-D Ig preparations, see E. Kenny-Walsh, for the Irish Hepatology Research Group, Clinical outcome after hepatitis C infection from contaminated anti-D immune globulin, N Engl J Med 340 (1999) 1228–1233, and 0.3% of children with hepatitis C, see M. Wiese, F. Berr, M. Lafrenz, H. Porst, V. Olsen, Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study, Hepatology 32 (2000) 91–96. These findings indicate that size and source of infection, age and gender are important variables affecting the course and outcome of chronic hepatitis C.
The treatment of patients with chronic HCV infection is based largely on consensus guidelines, see National Institutes of Health Consensus Development Conference Panel statement: management of hepatitis C. Hepatology 26 (1997) Suppl 1:2S–10S, and EASL International Consensus Conference on Hepatitis C: Paris, 26–28, Feb. 1999, consensus statement. J Hepatol 30 (1999) 956–961. The 1999 recommendations, see EASL International Consensus Conference on Hepatitis C: Paris, 26–28, Feb. 1999, consensus statement. J Hepatol 30 (1999) 956–961, suggest that naïve patients with the above-described indications and without contraindications to treatment with interferon or ribavirin should receive combination therapy. Treatment consists of 3 million U of interferon-α administered subsetaneously three times a week and 1200 mg of ribavirin orally per day for patients with weight greater than 75 kg and 1000 mg of ribavirin for those less than 75 kg. Usually, ribavirin is taken in divided doses, given in the morning and evening, and interferon is given before bedtime.
The efficiency of these therapies is usually and conventionally determined by measuring a biochemical response (normalization of alanine aminotransferase levels), but recently the introduction of assays for the detection of HCV RNA have allowed the assessment of virologic response (as defined by a negative result on a qualitative PCR assay for HCV RNA) as a criteria for successful therapy as well. Since responses to therapy may not be maintained after treatment is stopped, the success of clinical trials has been evaluated in terms of the response at the end of therapy (end-of-treatment response) and six months after the cessation of treatment (sustained treatment response). Patients with a sustained virologic response have a high probability of having a durable biochemical, virologic, and histologic response, see O. Reichard, H. Glaumann, A. Fryden, G. Norkrans, R. Wejstal, O. Weiland, Long-term follow-up of chronic hepatitis C patients with sustained virological response to alpha-interferon. J Hepatol 30 (1999) 783–787.
The rate of end-of-treatment response of HCV patients to interferon monotherapy was as high as 40 percent, but the rate of sustained response is less than half of this, see T. Poynard, P. Marcellin, S. S. Lee, et al. Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 352 (1998) 1426–1432, and J. G. McHutchison, S. C. Gordon, E. R. Schiff, et al. Interferon alfa-2b alone or in combination with ribavirin as initital treatment for chronic hepatitis C. N Engl J Med 339 (1998) 1485–1492. This is especially true in persons infected with HCV genotype 1a or 1b, the most prevalent genotypes in the United States and western Europe. Two large, prospective trials, see T. Poynard, P. Marcellin, S. S. Lee, et al., Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 352 (1998) 1426–1432, and J. G. McHutchison, S. C. Gordon, E. R. Schiff, et al., Interferon alfa-2b alone or in combination with ribavirin as initital treatment for chronic hepatitis C. N Engl J Med 339 (1998) 1485–1492, demonstrated that the combination of interferon-α and ribavirin significantly elevates the percentage of naive patients who have a sustained virologic response, from 16% to 40%. Also, both studies showed that the treatment regimens with optimal clinical outcome were associated with the viral genotype and pre-treatment viral load. For patients infected with HCV genotype 2 or 3 and in those with low viral loads before treatment, the response was maximal after 24 weeks of the treatment, whereas patients infected with genotype 1 and those with a high viral load before treatment required a course of 48 weeks for an optimal outcome. This finding led to the recommendation that the duration of treatment should be based on the HCV genotype and the pretreatment viral load, see EASL International Consensus Conference on Hepatitis C: Paris, 26–28, Feb. 1999, consensus statement. J Hepatol 30 (1999) 956–961. However, since tests for the quantification of HCV RNA are still not standardized, and since the viral load naturally fluctuates over time, the viral load is currently not routinely used for determining the treatment regimen.
Therapy of chronic hepatitis C has greatly improved in recent years with the use of interferon-α and ribavirin combined therapy. The therapy has been further improved more recently with the use of pegylated interferons (PEG-IFNs), again combined with ribavirin. The recent NIH Consensus Conference of the Management of Hepatitis C has concluded that on the basis of available data the highest response rates to antiviral therapy for the treatment of chronic hepatitis C have been achieved using the combination of PEG-IFNs and ribavirin, at least for patients infected with HCV-1 and such regimen has been therefore proposed as the new standard of therapy for chronic hepatitis, see C A. Alberti and L. Benvegnu, Management of hepatitis C, J. Hepatology 38 (2003) S104–S118.
Factors influencing the rate of sustained virological response include viral and host factors, as well as the pathogenesis state of the liver. The viral factors include viral genotype (types 1a, 2 and 3 are favorable to response), level of viraemia (less than 2 million copies/ml is favorable), and level of viral heterogeneity (degree of variability in E2/NS1 region of HCV correlates with response to IFN). The favorable host factors include younger age (less than 40) as well as female sex. The beneficial pathogenic factors are lower ALT and AST levels before treatment, absence of cirrhosis and low fibrotic histological scores, and lower hepatic iron content, see A. Alberti and L. Benvegnu, Management of hepatitis C, J. Hepatology 38 (2003) S104–S118, and G. M. Lauer and B. D. Walker, Hepatitis C virus infection, N Engl J Med 345 (2001) 41–52. In addition, due to the great extent of inter-individual variations in response to the treatment, it has been speculated that host genetic factors may also play an important role.
CD81 is a membrane bound protein composed of four transmembrane and two extracellular domains with a molecular weight of 26-kDa, see S. Levy, S. C. Todd, and H. T. Maecker, CD81 (TAPA-1): A molecule involved in signal transduction and cell adhesion in the immune system. Annu. Rev. Immunol. 16 (1998) 89–109. It is a member of the superfamily of proteins known as tetraspanins, see H. T. Maecker, S. C. Todd, and S. Levy, The tetraspanin superfamily: molecular facilitators. FASEB J. 11 (1997) 428–442. Most tetraspanins were originally identified as leukocyte antigens; however, it is now becoming evident that generally tetraspanins, particularly CD81, is expressed in many different cell types and involved in a variety of cellular functions including cell adhesion and migration, alteration of cell morphology, and activation state of a cell, see I. Tachibana and M. E. Hemler, Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance, J. Cell Biol. 146 (1999) 893–904. In the immune system, on the B cell CD81 forms a complex with CD21, CD19, and Leu13. Formation of this complex decreases the threshold for B cell activation through the B cell receptor by bridging Ag specific recognition and CD21-mediated complement recognition, see D. T. Fearon and R. H. Carter, The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13 (1995) 127–149. On T cells CD81 associates with CD4 and CD8 and provides a costimulatory signal with CD3, see T. Imai, M. Kakizaki, M. Nishimura, and O. Yoshie, Molecular analyses if the association of CD4 with two members of the transmembrane 4 superfamily, CD81 and CD82. J. Immunol. 15 (1995) 1229–1239. It is also shown that expression of CD81 by T cells greatly enhances cognate T-B cell interactions and greatly amplifies Th2 polarized intracellular activation pathways, see J. Deng, R. H. Dekruyff, G. J. Freeman, D. T. Umetsu, and S. Levy, Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses, Intl. Immunol. 14 (2002) 513–523.
Recently, it was shown that HCV particles bind CD81 and this binding is mediated by the interaction of the second extracellular loop of CD81 with HCV envelope 2 glycoprotien in vitro, see P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Binding of hepatitis C virus to CD81. Science 282 (1998) 938–941. Since the interaction between CD81 and E2 is sufficient for binding of whole HCV particle, it was postulated that CD81 may act as a receptor for the attachment and entrance of HCV into the cell, see M. Flint, C. Maidens, L. D. Loomis-Price, C. Shotton, J. Dubuisson, P. Monk, A. Higginbottom, S Levy, and J. A. McKeating, Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor CD81. J. Virol. 73 (1999) 6235–6244. However, until now there is no conclusive evidence to demonstrate the involvement of CD81 in the cellular uptake of HCV virions. More recently, it was showed that the protein level of membrane bound CD81 in isolated human peripheral blood cells and hepatocytes was significantly down-regulated by the treatment of interferon-α alone or combined with ribavirin, see B. Kronenberger, B. Ruster, R. Liez, S. Weber, A. Piier, J. H. Lee, W. K. Roth, and S. Zeuzem, Interferon alfa down-regulates CD81 in patients with chronic hepatitis C. Hepatology 33 (2001) 1518–1526. Also, levels of total CD81 protein of the PBLs of HCV-infected patients are significantly higher than those of the healthy subjects. Furthermore, cell surface-associated CD81 protein was lower 4 weeks after initiation of therapy in patients with an initial virologic response compared with initial virologic non-responders. Therefore, it is concluded that interferon-α and ribavirin regulate the expression of CD81 in vitro and in vivo. CD81 expression correlates with initial virologic response in patients with HCV infection. However, the detailed regulatory mechanism of CD 81 expression by interferon-α and ribavirin remains unclear. Moreover, the underlying reason that differentiates patients with different level of CD81 expression in response to therapy requires further studies at the genomic level, particularly for the sustained response of the therapy.